PubMed Health⌕ Search

Biomedical subjects

K Chapman

Publications and source records attributed to K Chapman.

At least 19 recordsLinked to original sources
PubMed1998

Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2.

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 microgram/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054, 522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.

Animals↗
PubMed1998

Selective interactions between prostate fibroblast and epithelial cells in co-culture maintain the BPH phenotype.

Paracrine interactions between primary cultured prostate epithelial cells and stromal fibroblasts were investigated in relation to morphology, growth, androgen sensitivity and secretory activities using co-cultures in which the two populations were separated by a microporous membrane. In this new model system, both cell types maintained several aspects of the differentiated phenotype including the capacity to express 5alpha-reductase iso-enzymes and androgen receptors, to respond to androgens and to secrete prostate-specific antigen by the epithelial cells. Morphological studies demonstrated that the cells grown in co-culture exhibited round nuclei, tonofibrils and microvilli in epithelial cells and elongated nuclei, large amounts of Golgi apparatus and cilia in the fibroblasts, all indicative of the differentiated state. The co-culture system highlights the importance of the metabolic co-operation between prostate fibroblast and epithelial cells for preserving the phenotypic characteristics associated with the human prostate in vivo.

Cell Communication↗
PubMed1998

A novel coculture model for benign prostatic hyperplasia expressing both isoforms of 5 alpha-reductase.

We have developed a coculture system for primary fibroblast and epithelial cells derived from benign prostatic hyperplasia (BPH) that retained many of the characteristics of the intact human prostate. In contrast to separately cultured prostate fibroblast and epithelial cells, cocultures of fibroblasts and epithelial cells maintained messenger ribonucleic acid expression and functional activity for both isoenzymes of 5 alpha-reductase (type I and type II) as well as maintained expression of androgen receptors and prostate-specific antigen. Furthermore, levels of prostate-specific antigen secreted by cocultured epithelial cells were increased by treatment with androgens, mimicking the situation in the human gland. This contrasted with conventionally cultured fibroblasts or epithelial cells, which failed to express 50 alpha-reductase type II and rapidly lost expression of androgen receptors and androgen sensitivity upon being placed into culture. Electron microscopy demonstrated intracellular structures indicative of the differentiated state of the cocultured cell types, including round nuclei, tonofibrils, and microvilli in epithelial cells and elongated nuclei; large amounts of Golgi and cilia; along with immature collagen fibers in fibroblasts. The present study demonstrates that the coculture model reflects more closely the in vivo system for human BPH and is thus a far more suitable model for investigating the molecular and cellular events that underlie BPH than current in vitro systems.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

The localisation and expression of 5 alpha-reductase types I and II mRNAs in human hyperplastic prostate and in prostate primary cultures.

The expression and localisation of mRNAs for 5 alpha reductase Type I (5 alpha R-I) and Type II (5 alpha R-II) isoenzymes in human benign prostatic hyperplasia (BPH) were investigated by RT-PCR and by in mini hybridisation (ISH) using digoxigenin labelled riboprobes. In addition, we also examined the isoenzymes mRNA expression in primary BPH cultures of separated stroma/fibroblast and epithelial cells to determine whether primary cultures are appropriate models in which to investigate 5 alpha R activity and regulation. The results demonstrated conclusively the presence of mRNA encoding both isoenzymes in all specimens so far examined. Additionally, the presence of a functional 5 alpha R-I and -II activity in BPH was confirmed by enzyme assays. ISH studies localised the mRNA expression to both the fibroblast/stromal component as well as the epithelial cells of the hyperplastic tissue. In the glandular regions the expression for both isoenzymes was particularly strong in the basal layers of the epithelium whereas mRNA expression in the secretory cells was less pronounced. Expression of 5 alpha R-I and -II mRNAs in fibroblast was on the other hand variable with high expression in some areas and little in others. These findings were supported by our primary culture experiments which demonstrated that both the fibroblast and epithelial cells maintain a capacity to express both isoenzymes in vitro. In the case of the fibroblast, the capacity to express the isoenzymes was maintained following the sequential passaging of the cells up to passage 6, after which the cells no longer expressed either isoenzyme.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Effects of the long acting beta agonist formoterol on asthma control in asthmatic patients using inhaled corticosteroids. The Netherlands and Canadian Formoterol Study Investigators.

BACKGROUND: The long acting beta 2 agonist formoterol has proved to be an effective bronchodilator with a prolonged action of 12-14 hours. However, the precise role of formoterol in the maintenance treatment of asthma is still under debate. A study was performed to investigate the efficacy and safety of treatment with formoterol for six months in subjects with asthma. METHODS: In a multicentre double blind, placebo controlled, parallel group study 239 subjects with mild to moderate asthma were randomly assigned to treatment with either inhaled formoterol 24 micrograms twice daily (n = 125) or placebo (n = 114) during eight months. The study consisted of a four week run in period, a 24 week treatment period, and a four week washout period. All subjects were using regular inhaled corticosteroids (100-3200 micrograms daily) but were still needing at least five inhalations of short acting beta 2 agonist per week for symptom relief. The study was performed in 10 outpatient clinics in Canada, and five outpatient clinics and one coordinating centre for 44 Dutch general practitioners in The Netherlands. Twice daily self-reported peak expiratory flow (PEF) measurements, symptom scores, and rescue beta 2 agonist use during the last 28 treatment days compared with baseline values were used as main outcome measures. Spirometric values were measured at entry, at the start of treatment, after four, 12 and 24 weeks of treatment, and after four weeks washout. RESULTS: One hundred and twenty five subjects received formoterol 24 micrograms twice daily via Turbohaler and 114 received placebo. Baseline FEV1 was 67.1% predicted and mean bronchodilator reversibility was 26%. The mean total asthma symptom score was 3.6 (maximum possible 21). A significant decrease in symptoms in favour of formoterol (difference from placebo -0.64, 95% CI -0.04 to -1.23, p = 0.04) was observed. Compared with placebo, morning PEF increased (difference from placebo 28 l/min, 95% CI 18.3 to 37.7, p = 0.0001) and the use of short acting beta 1 agonists decreased (daytime difference from placebo -1.1 inhalation, 95% CI -1.4 to -0.7, p = 0.0001) in the formoterol group. PEF returned to baseline following discontinuation of formoterol, as did asthma symptom scores. Thirty three patients treated with formoterol and 32 treated with placebo required treatment with prednisolone during the study (58 and 55 courses, respectively). CONCLUSIONS: Adding formoterol 24 micrograms twice daily by Turbohaler to inhaled corticosteroids was effective in improving symptom scores and morning PEF, and decreasing the use of rescue beta 2 agonists. There was no apparent loss of asthma control during 24 weeks of treatment with formoterol.

Adrenergic beta-Agonists↗
PubMed1996

A randomized and controlled trial of hydrotherapy in rheumatoid arthritis.

OBJECTIVE: The aim of this study was to evaluate the therapeutic effects of hydrotherapy which combines elements of warm water immersion and exercise. It was predicted that hydrotherapy would result in a greater therapeutic benefit than either of these components separately. METHODS: One hundred thirty-nine patients with chronic rheumatoid arthritis were randomly assigned to hydrotherapy, seated immersion, land exercise, or progressive relaxation. Patients attended 30-minute sessions twice weekly for 4 weeks. Physical and psychological measures were completed before and after intervention, and at a 3-month followup. RESULTS: All patients improved physically and emotionally, as assessed by the Arthritis Impact Measurement Scales 2 questionnaire. Belief that pain was controlled by chance happenings decreased, signifying improvement. In addition, hydrotherapy patients showed significantly greater improvement in joint tenderness and in knee range of movement (women only). At followup, hydrotherapy patients maintained the improvement in emotional and psychological state. CONCLUSIONS: Although all patients experienced some benefit, hydrotherapy produced the greatest improvements. This study, therefore, provides some justification for the continued use of hydrotherapy.

Arthritis, Rheumatoid↗
PubMed1996

Genetic and biochemical dissection of transgenic RNA-mediated virus resistance.

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.

Capsid↗
PubMed1995

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

Amino Acid Sequence↗
PubMed1995

p53-mediated transcriptional activity increases in differentiating epidermal keratinocytes in association with decreased p53 protein.

The regulation of p53 protein synthesis and p53-mediated gene transactivation were evaluated in cultured mouse keratinocytes maintained as basal cells or induced to differentiate by Ca2+ > 0.1 mM. p53 protein half-life, p53 protein synthesis and the level of p53 mRNA decreased during terminal differentiation, as detected by immunoprecipitation with a panel of p53-specific antibodies and Northern blotting. Thus differentiating keratinocytes have lower levels of p53 protein. This decline is not observed following growth arrest alone, or in papilloma cell lines which do not terminally differentiate in response to Ca2+. In contrast, the ability of endogenous p53 to transactivate transcription from the PG13 CAT plasmid increased during differentiation in vitro. This change in activity cannot be explained by changes in p53 conformation or nuclear localization. Consistent with these findings, mRNA for the p53-mediated genes WAF1 and mdm-2 increased with Ca(2+)-induced differentiation in a time dependent manner, suggesting activation of p53 contributes to the differentiated phenotype. However, p53-null mice exhibit histologically normal skin and epidermal keratinocytes from these mice express the appropriate markers of differentiation and suppression of DNA synthesis in vitro when the [Ca2+] is > 0.1 mM. The observation that proliferating cells have higher levels of p53 protein which is less active for its function than differentiated cell types could have a consequence for the selection of p53 gene mutations during carcinogenesis, depending upon the stage of differentiation of the tumor cell type.

Animals↗
PubMed1995

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.

Aggrecans↗
PubMed1995

In vivo effects of stromelysin on the composition and physical properties of rabbit articular cartilage in the presence and absence of a synthetic inhibitor.

OBJECTIVE: To characterize the effects of intraarticular injection of recombinant human stromelysin (SLN) on the matrix composition and physical properties of cartilage from lapine stifle joints and the modulation of these effects by the systemic administration of an N-carboxyalkyl synthetic matrix metalloproteinase inhibitor, L-696,418. METHODS: Female 6-8-week-old New Zealand white rabbits received an intraarticular injection of 100 micrograms activated SLN in 1 stifle joint and buffer in the contralateral control knee; these animals were killed after 1 hour. A separate group of animals received an intravenous injection of either 30 mg/kg L-696,418 or buffer prior to intraarticular injection of SLN. Joints were dissected and analyzed for proteoglycan (PG) loss into joint fluid, tissue biochemical composition, and histology by toluidine blue or anti-VDIPEN antibody staining, or were frozen for physical property analysis. Disks of femoropatellar groove cartilage were harvested from the stifle joint and tested in uniaxially confined compression for determination of electromechanical and mechanical properties. RESULTS: Lapine stifle joints that received injection of SLN without systemic administration of L-696,418 showed a 13-fold increase in loss of PG into synovial fluid. Cartilage from these joints showed significant decreases in streaming potential at 1 Hz and electrokinetic coupling coefficient, but no change in equilibrium modulus, dynamic stiffness, or hydraulic permeability. Systemic treatment with L-696,418 resulted in a significant decrease in loss of PG into joint fluid and elimination of changes in cartilage high-frequency streaming potential and coupling coefficient in joints that were injected with SLN. CONCLUSION: The 1-hour exposure to SLN in vivo resulted in loss of PG and exposure of the VDIPEN epitope of the aggrecan core protein in the superficial region of the tissue near the articular surface. This highly localized degradation resulted in electromechanical behavior changes, but little or no change occurred in mechanical properties. Systemic administration of L-696,418 significantly decreased loss of PG from cartilage and prevented the highly localized tissue degradation and the resultant changes in electromechanical behavior caused by intraarticular SLN injection.

Animals↗
PubMed1995

Testing patients for HIV before surgery: the views of doctors performing surgery.

The aim of this study was to investigate the attitudes of doctors performing surgery to the HIV antibody testing of surgical patients. Fifty of eighty (62.5%) doctors performing surgery who are working in two London teaching hospitals returned completed anonymous postal questionnaires. Sixty-six per cent of the sample would like some form of compulsory testing of pre-operative patients, although most of them feel that this is only necessary for patients considered to be in 'high-risk groups'. Eighty-four per cent believe that this would ensure their safety from infection during surgery. Forty-eight per cent agreed with testing patients without their consent. Results suggest that most of the doctors performing surgery in this study agree with compulsory HIV antibody testing of pre-operative patients in the belief that this would protect them from infection during surgery. The problems associated with compulsory testing and relying on such testing in order to protect doctors from infection during surgery are discussed.

AIDS Serodiagnosis↗
PubMed1994

The rat preprotachykinin-A promoter is regulated in PC12 cells by the synergistic action of multiple stimuli.

We demonstrate in PC12 cells that although nerve growth factor, forskolin or potassium-evoked depolarisation independently induced minimal or no expression from the rat preprotachykinin-A gene (rPPT) promoter linked to a reporter gene, exposure of the cells to various combinations of these stimuli specifically activated the rPPT promoter in transient transfection assays.

Animals↗
PubMed1994

A second generation of non-peptide cholecystokinin receptor antagonists and their possible therapeutic potential.

The profile of an acidic series of benzodiazepine CCK-B receptor antagonists is described. The tetrazolyl urea derivative L-368,935 had high affinity (CCK-B IC50 0.1 nM) and was one of the most selective (CCK-B/CCK-A 10,000) CCK-B antagonists known. L-368,935 was a CCK-B antagonist with high affinity on the rat ventromedial hypothalamic slice preparation (Kb 0.6 nM) and also blocked pentagastrin-induced calcium mobilization in GH3 cells. L-368,935 had potent in vivo activity and antagonized pentagastrin-induced gastric acid secretion in the anesthetized rat and CCK-8S-induced aspartate release using microdialysis in the striatum of conscious rats. Activity within the central nervous system was confirmed by a mouse ex vivo binding assay and by direct measurement of the compound within the central nervous system using an HPLC assay. A second generation of CCK-B receptor antagonists such as L-368,935 will be important in determining the therapeutic potential of this class of compound in man.

Animals↗